The human fibroblast activation protein a

The human fibroblast activation protein (FAP) defined by monoclonal antibody (MAb) F19 is a cell surface antigen expressed in reactive stromal fibroblasts of breast, colorectal, lung and other epithelial cancers. In contrast to its stroma-specific localization in epithelial neoplasms, FAP is expressed in the malignant mesenchymal cells of bone and soft tissue sarcomas. FAP is transiently expressed in some fetal mesenchymal tissues but is absent or expressed at low levels in most adult tissues. FAP is induced in cultured fibroblasts and, in these cells, consists of a M(r) 95,000 subunit (FAP alpha) carrying the F19 epitope and a non-covalently bound M(r) 105,000 subunit (FAP beta) lacking the F19 epitope. Using MAb F19 and 5 newly derived MAbs, we identify 3 distinct epitopes on FAP alpha and tentatively assign one epitope to FAP beta. Analysis of detergent extracts of a FAP alpha high beta- sarcoma cell line by size exclusion-high performance liquid chromatography (HPLC) revealed that FAP alpha does not elute as a M(r) 95,000 species but as part of a high-molecular weight complex (M(r) > 400,000) that dissociates into M(r) 95,000 subunits in SDS gels. Immunoaffinity purification of FAP alpha followed by tryptic digestion, reversed-phase HPLC and microsequencing identified 3 unique FAP alpha peptides, with 2 showing sequence similarity (23/38 identical amino acids) to segments of CD26, a T-cell activation antigen. CD26 is a membrane-bound enzyme (dipeptidyl aminopeptidase IV), but immunopurified FAP alpha has little if any dipeptidase activity with typical CD26 substrates. Finally, studies with FAPlow leptomeningeal fibroblasts revealed that transforming growth factor-beta, 12-O-tetradecanoyl phorbol-13-acetate and retinoids can upregulate FAP expression, whereas serum and several other factors had no or little effect on FAP levels. FAP and CD26 may belong to a family of structurally related but functionally distinct activation proteins that are expressed on different cell types and show unique modes of regulation in normal and malignant cells.

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We have selected GD3-deficient human melanoma cell lines, in order to investigate the function of GD3 ganglioside. This was done by treating SK-MEL-28 cells with anti-GD3 antibody (R24) and rabbit complement and subsequent subcloning of the surviving cells, resulting in the derivation of two cell lines deficient in the cell surface expression of GD3. Neither cell line (designated SK-MEL-28-N1 and SK-MEL-28-N2) had detectable cell surface expression of GD3 as analyzed with monoclonal antibody R24, and no GD3 was detectable in either cell line by glycolipid isolation, thin-layer chromatography, or resorcinol-HC1 spray, but thin-layer chromatography immunostaining with monoclonal antibody R24 showed the presence of low amounts of GD3 in both N1 and N2 (1/40 of the amount in the parent cell line in N1 and 1/500 in N2). In SK-MEL-28-N1, the residual GD3 was shown by immunofluorescence assays on permeabilized cells to be present in discrete intracellular organelles, suggesting that these cells have a defect in the transport of GD3 as well as in its synthesis. Both SK-MEL-28-N1 and -N2 had an increase in detectable GM3 expression. The mutant cell lines had altered cell morphology in comparison to the parent cell line and both had slower growth rates in vitro and lower tumorgenicity in nu/nu mice. These results indicate that GD3 ganglioside plays an important role in proliferation and growth of melanoma cells.

Oncogenic transformation has been associated with decreased fibronectin (FN) matrix assembly. For example, both the HT-1080 fibrosarcoma and MAT-LyLu cell lines fail to assemble a FN matrix when grown in monolayer culture (2-dimensional [2D] system). In this study, we show that these cells regain the ability to assemble a FN matrix when they are grown as aggregates (3-dimensional [3D] system). FN matrix assembly in 3D correlates with decreased Raf-1 protein expression compared with cells grown in monolayer culture. This effect is associated with reduced Raf-1 mRNA levels as determined by quantitative RT-PCR and not proteasome-mediated degradation of endogenous Raf-1. Interestingly, transient expression of a Raf-1 promoter-reporter construct demonstrates increased Raf-1 promoter activity in 3D, suggesting that the transition to 3D culture may modulate Raf-1 mRNA stability. Finally, to confirm that decreased Raf-1 expression results in increased FN matrix assembly, we used both pharmacological and small interfering RNA knockdown of Raf-1. This restored the ability of cells in 2D culture to assemble a FN matrix. Moreover, overexpression of Raf-1 prevented FN matrix assembly by cells cultured in 3D, resulting in decreased aggregate compaction. This work provides new insight into how the cell microenvironment may influence Raf-1 expression to modulate cell-FN interactions in 3D. INTRODUCTIONFibronectin (FN) is a multifunctional, adhesive glycoprotein that has wide tissue distribution and is essential for normal development and tissue repair (Hynes, 1990;Schwarzbauer, 1991;Sottile and Hocking, 2002). Cells secrete FN as a disulfide-bonded dimer that binds principally to integrin cell surface receptors. Integrin-FN interactions allow unfolding of the soluble protein and its assembly into a detergentinsoluble fibrillar matrix that can modulate cell morphology, growth, and tissue architecture (Schwarzbauer and Sechler, 1999;Wierzbicka-Patynowski and Schwarzbauer, 2003). FN matrix assembly can also regulate the subsequent deposition and organization of other extracellular matrix molecules, including fibrinogen, collagen-1, and thrombospondin-1 (Sottile and Hocking, 2002). As a consequence, FN fibrillogenesis initiates the formation of a dynamic protein meshwork that provides important structural and environmental cues required for normal cell behavior.One hallmark of malignant transformation in vitro is the loss of FN matrix assembly in two-dimensional (2D) culture. For example, transformed cells frequently show decreased FN synthesis, loss of FN receptor expression, or both (Olden and Yamada, 1977;Plantefaber and Hynes, 1989), and in many cases, the loss of surface FN assayed in these cells correlates with malignant transformation, in vivo. Similarly, oncogenic cells can demonstrate loss of normal integrin function, despite adequate receptor expression. For example, human HT-1080 fibrosarcoma cells express the FN-binding integrin ␣5␤1 and adhere to FN-coated substrates, but they lack the ability to assemble a FN matrix e...

Most human neuroblastoma tumors are characterized by the high expression of GD2 (or GD2 and/or GM2) gangliosides, whereas melanomas characteristically express GD3 ganglioside. The molecular basis for these patterns was investigated by examining the relationship between ganglioside levels, glycosyltransferase (GM2/GD2 synthase and GD3 synthase) activity, and corresponding mRNA levels in a panel of human neuroblastoma and melanoma cell lines. In general, the ganglioside patterns could be explained by the levels of the transferases and their mRNA, indicating control at the level of transcription. A key role was noted for GD3 synthase. Notably, it was found that neuroblastoma cell lines with high GD2 ganglioside levels had low levels of GD3, its synthase, and mRNA for the enzyme even though this step provides the substrate for GD2 synthesis. The key role for GD3 synthase was also examined by stably transfecting GD3 synthase cDNA into a neuroblastoma cell line (SH-SY5Y) not expressing GD3 and GD2. The resulting cell line had high levels of GD2 ganglioside and altered morphology and growth characteristics.

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